Identification of Nicotiana tabacum Linkage Group Corresponding to the Q Chromosome Gene(s) Involved in Hybrid Lethality

BACKGROUND: A linkage map consisting of 24 linkage groups has been constructed using simple sequence repeat (SSR) markers in Nicotiana tabacum. However, chromosomal assignments of all linkage groups have not yet been made. The Q chromosome in N. tabacum encodes a gene or genes triggering hybrid lethality, a phenomenon that causes death of hybrids derived from some crosses. METHODOLOGY/PRINCIPAL FINDINGS: We identified a linkage group corresponding to the Q chromosome using an interspecific cross between an N. tabacum monosomic line lacking the Q chromosome and N. africana. N. ingulba yielded inviable hybrids after crossing with N. tabacum. SSR markers on the identified linkage group were used to analyze hybrid lethality in this cross. The results implied that one or more genes on the Q chromosome are responsible for hybrid lethality in this cross. Furthermore, the gene(s) responsible for hybrid lethality in the cross N. tabacum × N. africana appear to be on the region of the Q chromosome to which SSR markers PT30342 and PT30365 map. CONCLUSIONS/SIGNIFICANCE: Linkage group 11 corresponded to the Q chromosome. We propose a new method to correlate linkage groups with chromosomes in N. tabacum

Genes in S and T Subgenomes Are Responsible for Hybrid Lethality in Interspecific Hybrids between Nicotiana tabacum and Nicotiana occidentalis

Many species of Nicotiana section Suaveolentes produce inviable F(1) hybrids after crossing with Nicotiana tabacum (genome constitution SSTT), a phenomenon that is often called hybrid lethality. Through crosses with monosomic lines of N. tabacum lacking a Q chromosome, we previously determined that hybrid lethality is caused by interaction between gene(s) on the Q chromosome belonging to the S subgenome of N. tabacum and gene(s) in Suaveolentes species. Here, we examined if hybrid seedlings from the cross N. occidentalis (section Suaveolentes)×N. tabacum are inviable despite a lack of the Q chromosome.Hybrid lethality in the cross of N. occidentalis×N. tabacum was characterized by shoots with fading color. This symptom differed from what has been previously observed in lethal crosses between many species in section Suaveolentes and N. tabacum. In crosses of monosomic N. tabacum plants lacking the Q chromosome with N. occidentalis, hybrid lethality was observed in hybrid seedlings either lacking or possessing the Q chromosome. N. occidentalis was then crossed with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT), to reveal which subgenome of N. tabacum contains gene(s) responsible for hybrid lethality. Hybrid seedlings from the crosses N. occidentalis×N. tomentosiformis and N. occidentalis×N. sylvestris were inviable.Although the specific symptoms of hybrid lethality in the cross N. occidentalis×N. tabacum were similar to those appearing in hybrids from the cross N. occidentalis×N. tomentosiformis, genes in both the S and T subgenomes of N. tabacum appear responsible for hybrid lethality in crosses with N. occidentalis

Species origin of genomic factors in Nicotiana nudicaulis Watson controlling hybrid lethality in interspecific hybrids between N. nudicaulis Watson and N. tabacum L.

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis × N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris × N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris × N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla × N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla × N. tabacum and N. sylvestris × N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis

Histograms indicating hybridity for hybrid seedlings of <i>N. trigonophylla</i>×<i>N. tabacum</i>.

<p>The DNA content of 16,000–22,000 nuclei was determined by flow cytometry. (A) A sample of leaf cells from <i>N. trigonophylla</i> as the internal standard. The two peaks are characteristic of G1 nuclei and G2/M nuclei. (B) The G1 peak of a viable hybrid plant was intermediate between the two parents. The abbreviations <i>N. tri</i> and <i>N. tab</i> are for <i>N. trigonophylla</i> and <i>N. tabacum,</i> respectively. (C, D and E) The G1 peaks of vitrified hybrid seedlings were also intermediate between parents. However, the DAPI fluorescence values of these G1 peaks were closer to that of <i>N. trigonophylla</i> and the fluorescence values of G1 peaks differed among vitrified hybrid plants. Furthermore, subG1 peaks (black arrows), indicating nuclear fragmentation, with lower fluorescence values than that of G1 peaks appeared.</p

A hybrid of <i>N. trigonophylla</i>×<i>N. tabacum</i>.

<p>(A) Shape of a hybrid plant that grew to maturity and flowered. (B) Leaves of <i>N. trigonophylla</i>, a hybrid and <i>N. tabacum</i> (left to right). (C and D) Flowers of <i>N. trigonophylla</i>, a hybrid and <i>N. tabacum</i> (left to right). (E) Image of root tip cell of a hybrid plant, showing the number of chromosomes. (F) Confirmation of hybrid formation by RAPD analysis using primer OPE-14. The bands characteristic of both parents are indicated by arrows. M, size marker OneSTEP Ladder 1kb (1–10 kbp). Scale bars are 10 cm (A), 3 cm (B), 1 cm (C and D) and 100 µm (E).</p

Viability of hybrid seedlings generated from crosses between <i>N. occidentalis</i> and two progenitors of <i>N. tabacum</i> at different temperatures.

a<p>Type II, browning of hypocotyl and roots; Type V, fading of shoot color.</p

Conventional crossing between <i>N. occidentalis</i> and two progenitors of <i>N. tabacum</i>.

<p>Conventional crossing between <i>N. occidentalis</i> and two progenitors of <i>N. tabacum</i>.</p

A hybrid of <i>N. sylvestris</i>×<i>N. trigonophylla</i>.

<p>(A) Shape of a hybrid plant that grew to maturity and flowered. (B) Leaves of <i>N. trigonophylla</i>, a hybrid and <i>N. tabacum</i> (left to right). (C and D) Flowers of <i>N. trigonophylla</i>, a hybrid and <i>N. tabacum</i> (left to right). (E) Image of root tip cell of a hybrid plant, showing the number of chromosomes. (F) Confirmation of hybrid formation by RAPD analysis using primer OPF-03. The bands characteristic of both parents are indicated by arrows. M, size marker OneSTEP Ladder 1kb (1–10 kbp). Scale bars are 10 cm (A), 3 cm (B), 1 cm (C and D) and 100 µm (E).</p

Several types of abnormal hybrid plants from <i>N. trigonophylla</i>×<i>N. tabacum</i> cultured at 28°C.

<p>(A) A vitrified hybrid plant. (B) A vitrified hybrid plant that changed from a browning tumorous plant. (C) A vitrified hybrid plant coexisting with tumors. (D) Vitrification that occurred on a tumorous hybrid plant. (E) A vitrified hybrid plant that changed from a tumorous plant, which had developed at the crown of a malformed plant. (F) A tumorous hybrid plant that died 10 DAG. (G) A malformed hybrid plant that died 14 DAG. (H) A viable hybrid plant coexisting with tumors. (I) A tumorous hybrid plant that developed at the crown of a yellowing plant. (J) A tumorous hybrid plant that developed at the crown of a browning vitrified plant. (K) A malformed hybrid plant. Scale bars are 1 mm.</p

Interspecific hybridization of <i>N. trigonophylla</i>×<i>N. tabacum</i>.

<p>Interspecific hybridization of <i>N. trigonophylla</i>×<i>N. tabacum</i>.</p